Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

Neutrophil extracellular trap formation and gene programs distinguish TST/IGRA sensitization outcomes among Mycobacterium tuberculosis exposed persons living with HIV.

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and show no evidence of immune sensitization to Mycobacterium tuberculosis (Mtb) as defined by persistently negative tuberculin skin tests (TST) and interferon gamma release assays (IGRA). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophil Mtb infection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected with Mtb H37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. Since there was no significant differential transcriptional response at 1h between infected PMNHITTIN and PMNHIT, we focused on the 6h timepoint. When compared to uninfected PMN, PMNHITTIN displayed 3106 significantly upregulated and 3548 significantly downregulated differentially expressed genes (DEGs) (absolute cutoff of a log2FC of 0.2, FDR < 0.05) whereas PMNHIT demonstrated 3816 significantly upregulated and 3794 significantly downregulated DEGs following 6h Mtb infection. Contrasting the log2FC 6h infection response to Mtb from PMNHITTIN against PMNHIT, 2285 genes showed significant differential response between the two groups. Overall PMNHITTIN had a lower fold change response to Mtb infection compared to PMNHIT. According to pathway enrichment, Apoptosis and NETosis were differentially regulated between HITTIN and HIT PMN responses after 6h Mtb infection. To corroborate the blunted NETosis transcriptional response measured among HITTIN, fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTIN compared to PMNHIT, showing that PMNHITTIN have a distinct response to Mtb.

Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

Broadcasting of amplitude- and frequency-modulated c-di-GMP signals facilitates cooperative surface commitment in bacterial lineages.

Work on surface sensing in bacterial biofilms has focused on how cells transduce sensory input into cyclic diguanylate (c-di-GMP) signaling, low and high levels of which generally correlate with high-motility planktonic cells and low-motility biofilm cells, respectively. Using Granger causal inference methods, however, we find that single-cell c-di-GMP increases are not sufficient to imply surface commitment. Tracking entire lineages of cells from the progenitor cell onward reveals that c-di-GMP levels can exhibit increases but also undergo oscillations that can propagate across 10 to 20 generations, thereby encoding more complex instructions for community behavior. Principal component and factor analysis of lineage c-di-GMP data shows that surface commitment behavior correlates with three statistically independent composite features, which roughly correspond to mean c-di-GMP levels, c-di-GMP oscillation period, and surface motility. Surface commitment in young biofilms does not correlate to c-di-GMP increases alone but also to the emergence of high-frequency and small-amplitude modulation of elevated c-di-GMP signal along a lineage of cells. Using this framework, we dissect how increasing or decreasing signal transduction from wild-type levels, by varying the interaction strength between PilO, a component of a principal surface sensing appendage system, and SadC, a key hub diguanylate cyclase that synthesizes c-di-GMP, impacts frequency and amplitude modulation of c-di-GMP signals and cooperative surface commitment.

Utility of carboplatin therapeutic drug monitoring for the treatment of neonate and infant retinoblastoma patients in the United Kingdom.

BACKGROUND: Retinoblastoma is the most common intra-ocular malignancy in children and frequently presents in very young patients who commonly require intravenous carboplatin. Delivering this is challenging due to a lack of uniform dosing recommendations, rapid changes in physiological function and the risk of side-effects. METHODS: We conducted a retrospective review of neonates and infants in the UK with retinoblastoma, who have undergone carboplatin therapeutic drug monitoring (TDM). We report on the pharmacokinetic, treatment efficacy and toxicity data. RESULTS: In total, 29 patients (median age 5 weeks at treatment onset) underwent a total of 74 TDM guided cycles of chemotherapy, involving real time sampling and dose adjustment. An additional 13 patients underwent TDM sampling to modify doses between cycles. Without the adoption of TDM guided dosing, carboplatin exposures would have been ≥20% outside the target AUC in 38/78 (49%) of treatment cycles. Excellent responses and a reassuringly low incidence of toxicities were observed following dose adjustment, despite the young patient age and the implementation of dose increases in the majority of cases. CONCLUSIONS: Real time TDM is safe, effective and deliverable for neonates and infants receiving carboplatin for retinoblastoma and should be considered standard of care up to the age of 6 months.

Remote haemodynamic monitoring of pulmonary artery pressures in patients with chronic heart failure (MONITOR-HF): a randomised clinical trial.

BACKGROUND: The effect of haemodynamic monitoring of pulmonary artery pressure has predominantly been studied in the USA. There is a clear need for randomised trial data from patients treated with contemporary guideline-directed-medical-therapy with long-term follow-up in a different health-care system. METHODS: MONITOR-HF was an open-label, randomised trial, done in 25 centres in the Netherlands. Eligible patients had chronic heart failure of New York Heart Association class III and a previous heart failure hospitalisation, irrespective of ejection fraction. Patients were randomly assigned (1:1) to haemodynamic monitoring (CardioMEMS-HF system, Abbott Laboratories, Abbott Park, IL, USA) or standard care. All patients were scheduled to be seen by their clinician at 3 months and 6 months, and every 6 months thereafter, up to 48 months. The primary endpoint was the mean difference in the Kansas City Cardiomyopathy Questionnaire (KCCQ) overall summary score at 12 months. All analyses were by intention-to-treat. This trial was prospectively registered under the clinical trial registration number NTR7673 (NL7430) on the International Clinical Trials Registry Platform. FINDINGS: Between April 1, 2019, and Jan 14, 2022, we randomly assigned 348 patients to either the CardioMEMS-HF group (n=176 [51%]) or the control group (n=172 [49%]). The median age was 69 years (IQR 61-75) and median ejection fraction was 30% (23-40). The difference in mean change in KCCQ overall summary score at 12 months was 7·13 (95% CI 1·51-12·75; p=0·013) between groups (+7·05 in the CardioMEMS group, p=0·0014, and -0·08 in the standard care group, p=0·97). In the responder analysis, the odds ratio (OR) of an improvement of at least 5 points in KCCQ overall summary score was OR 1·69 (95% CI 1·01-2·83; p=0·046) and the OR of a deterioration of at least 5 points was 0·45 (0·26-0·77; p=0·0035) in the CardioMEMS-HF group compared with in the standard care group. The freedom of device-related or system-related complications and sensor failure were 97·7% and 98·8%, respectively. INTERPRETATION: Haemodynamic monitoring substantially improved quality of life and reduced heart failure hospitalisations in patients with moderate-to-severe heart failure treated according to contemporary guidelines. These findings contribute to the aggregate evidence for this technology and might have implications for guideline recommendations and implementation of remote pulmonary artery pressure monitoring. FUNDING: The Dutch Ministry of Health, Health Care Institute (Zorginstituut), and Abbott Laboratories.

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses.

Deposition of human amyloids is associated with complex human diseases such as Alzheimer's and Parkinson's. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro. The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.

Magnetic hybrid Pd/Fe-oxide nanoparticles meet the demands for ablative thermo-brachytherapy.

OBJECTIVE: To investigate the potential of hybrid Pd/Fe-oxide magnetic nanoparticles designed for thermo-brachytherapy of breast cancer, considering their specific loss power (SLP) and clinical constraints in the applied magnetic field. METHODS: Hybrid nanoparticles consisting of palladium-core and iron oxide shell of increasing thickness, were suspended in water and their SLPs were measured at varying magnetic fields (12-26 mT peak) and frequencies (50-730 kHz) with a commercial alternating magnetic field generator (magneTherm™ Digital, nanoTherics Ltd.). RESULTS: Validation of the heating device used in this study with commercial HyperMag-C nanoparticles showed a small deviation (±4%) over a period of 1 year, confirming the reliability of the method. The integration of dual thermometers, one in the center and one at the bottom of the sample vial, allowed monitoring of homogeneity of the sample suspensions. SLPs measurements on a series of nanoparticles of increasing sizes showed the highest heating for the diameter of 21 nm (SLP = 225 W/g) at the applied frequencies of 346 and 730 kHz. No heating was observed for the nanoparticles with the size <14 nm, confirming the importance of the size-parameter. The heating ability of the best performing Pd/Fe-oxide-21 was calculated to be sufficient to ablate tumors with a radius ±4 and 12 mm using 10 and 1 mg/mL nanoparticle concentration, respectively. CONCLUSIONS: Nanoparticles consisting of non-magnetic palladium-core and magnetic iron oxide shell are suitable for magnetic hyperthermia/thermal ablation under clinically safe conditions of 346 kHz and 19.1 mT, with minimal eddy current effects in combination with maximum SLP.

Interaction between the type 4 pili machinery and a diguanylate cyclase fine-tune c-di-GMP levels during early biofilm formation.

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.

PACAP is a pathogen-inducible resident antimicrobial neuropeptide affording rapid and contextual molecular host defense of the brain.

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

Ethnic minorities treated with new-generation drug-eluting coronary stents in two European randomised clinical trials.

BACKGROUND: Several ethnic minorities have an increased risk of cardiovascular events, but previous European trials that investigated clinical outcome after coronary stenting did not assess the patients' ethnic background. AIMS: To compare ethnic minority and Western European trial participants in terms of both cardiovascular risk profile and 1­year clinical outcome after percutaneous coronary intervention. METHODS: In the BIO-RESORT and BIONYX randomised trials, which assessed new-generation drug-eluting stents, information on patients' self-reported ethnic background was prospectively collected. Pooled patient-level data of 5803 patients, enrolled in the Netherlands and Belgium, were analysed in this prespecified analysis. The main endpoint was target vessel failure after 1 year. RESULTS: Patients were classified as belonging to an ethnic minority (n = 293, 5%) or of Western European origin (n = 5510, 95%). Follow-up data were available in 5772 of 5803 (99.5%) patients. Ethnic minority patients were younger, less often female, more often current smokers, more often medically treated for diabetes, and more often had a positive family history of coronary artery disease. The main endpoint target vessel failure did not differ between ethnic minority and Western European patients (3.5% vs 4.9%, hazard ratio 0.71, 95% confidence interval 0.38-1.33; p = 0.28). There was also no difference in mortality, myocardial infarction, and repeat revascularisation rates. CONCLUSIONS: Despite the unfavourable cardiovascular risk profile of ethnic minority patients, short-term clinical outcome after treatment with contemporary drug-eluting stents was highly similar to that in Western European patients. Further efforts should be made to ensure the enrolment of more ethnic minority patients in future coronary stent trials.

Structural studies of human fission protein FIS1 reveal a dynamic region important for GTPase DRP1 recruitment and mitochondrial fission.

Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal "arm" of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1. In human FIS1, NMR and X-ray structures show different arm conformations, but its importance for human DRP1 recruitment is unknown. Here, we use molecular dynamics simulations and comparisons to experimental NMR chemical shifts to show the human FIS1 arm can adopt an intramolecular conformation akin to that observed with yeast Fis1p. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human FIS1 with and without the arm. Using NMR, we observed the human FIS1 arm is also sensitive to environmental changes. We reveal the importance of these findings in cellular studies where removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission similar to the yeast system. Moreover, we determined that expression of mitophagy adapter TBC1D15 can partially rescue arm-less FIS1 in a manner reminiscent of expression of the adapter Mdv1p in yeast. These findings point to conserved features of FIS1 important for its activity in mitochondrial morphology. More generally, other tetratricopeptide repeat-containing proteins are flanked by disordered arms/tails, suggesting possible common regulatory mechanisms.

How Cell-Penetrating Peptides Behave Differently from Pore-Forming Peptides: Structure and Stability of Induced Transmembrane Pores.

Peptide-induced transmembrane pore formation is commonplace in biology. Examples of transmembrane pores include pores formed by antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) in bacterial membranes and eukaryotic membranes, respectively. In general, however, transmembrane pore formation depends on peptide sequences, lipid compositions, and intensive thermodynamic variables and is difficult to observe directly under realistic solution conditions, with structures that are challenging to measure directly. In contrast, the structure and phase behavior of peptide-lipid systems are relatively straightforward to map out experimentally for a broad range of conditions. Cubic phases are often observed in systems involving pore-forming peptides; however, it is not clear how the structural tendency to induce negative Gaussian curvature (NGC) in such phases is quantitatively related to the geometry of biological pores. Here, we leverage the theory of anisotropic inclusions and devise a facile method to estimate transmembrane pore sizes from geometric parameters of cubic phases measured from small-angle X-ray scattering (SAXS) and show that such estimates compare well with known pore sizes. Moreover, our model suggests that although AMPs can induce stable transmembrane pores for membranes with a broad range of conditions, pores formed by CPPs are highly labile, consistent with atomistic simulations.

Comparative study of cranial measurements between sexes from Brazil and The Netherlands: A cone-beam computed tomography study.

The objective of this study was to better understand human variation by comparing cone-beam computed tomography-based cranial measurements between both sexes of individuals from two distinct populations: Brazilian and Dutch. Cone-beam computed tomography volumes of 311 patients between 20 and 60 years from Brazil and The Netherlands were selected. Two radiologists performed 16 linear measurements in the maxillary sinuses and mandibular canal. Kruskall-Wallis test compared measurements of the two cranial structures between male and female for the two populations and four age ranges (20-30, 31-40, 41-50, 51-60). Mann-Whitney test compared individual measurements obtained from the cranial structures between male and female for each population, and between both populations for both sexes. Intra- and inter-observer reliability was assessed by intraclass correlation test (α = 0.05). No significant differences were found in the linear measurements among the experimental groups including sex, population and age group for both cranial structures (p > 0.05). Most of the cranial linear measurements were significantly higher for male than those for female irrespective of the population (p ≤ 0.05). When the populations were compared regardless of sex, Brazilians presented four significantly higher measurements, and Dutch presented seven significantly higher measurements (p ≤ 0.05). The assessed cranial structures did not differ between Brazilian and Dutch populations for both sexes and four age ranges. Multiple linear measurements differed between both populations with a predominance of larger dimensions for the Dutch population.

Using MRI to measure position and anatomy changes and assess their impact on the accuracy of hyperthermia treatment planning for cervical cancer.

PURPOSE: We studied the differences between planning and treatment position, their impact on the accuracy of hyperthermia treatment planning (HTP) predictions, and the relevance of including true treatment anatomy and position in HTP based on magnetic resonance (MR) images. MATERIALS AND METHODS: All volunteers were scanned with an MR-compatible hyperthermia device, including a filled waterbolus, to replicate the treatment setup. In the planning setup, the volunteers were scanned without the device to reproduce the imaging in the current HTP. First, we used rigid registration to investigate the patient position displacements between the planning and treatment setup. Second, we performed HTP for the planning anatomy at both positions and the treatment mimicking anatomy to study the effects of positioning and anatomy on the quality of the simulated hyperthermia treatment. Treatment quality was evaluated using SAR-based parameters. RESULTS: We found an average displacement of 2 cm between planning and treatment positions. These displacements caused average absolute differences of ∼12% for TC25 and 10.4%-15.9% in THQ. Furthermore, we found that including the accurate treatment position and anatomy in treatment planning led to an improvement of 2% in TC25 and 4.6%-10.6% in THQ. CONCLUSIONS: This study showed that precise patient position and anatomy are relevant since these affect the accuracy of HTP predictions. The major part of improved accuracy is related to implementing the correct position of the patient in the applicator. Hence, our study shows a clear incentive to accurately match the patient position in HTP with the actual treatment.

Application of the ESHO-QA guidelines for determining the performance of the LCA superficial hyperthermia heating system.

PURPOSE: This study aimed to assess the quality of the lucite cone applicator (LCA), the standard applicator for superficial hyperthermia at the Erasmus MC Cancer Institute, using the most recent quality assurance guidelines, thus verifying their feasibility. MATERIALS AND METHODS: The assessment was conducted on each of the six LCAs available for clinical treatments. The temperature distribution was evaluated using an infrared camera across different layers of a fat-muscle mimicking phantom. The maximum temperature increase, thermal effective penetration depth (TEPD), and thermal effective field size (TEFS) were used as quality metrics. The experimental results were validated through comparison with simulated results, using a canonical phantom model and a realistic phantom model segmented from CT imaging. RESULTS: A maximum temperature increase above 6 °C at 2 cm depth in the fat-muscle phantom for all the experiments was found. A mean negative difference between simulated and experimental data was of 1.3 °C when using the canonical phantom model. This value decreased to a mean negative difference of 0.4 °C when using the realistic model. Simulated and measured TEPD showed good agreement for both in silico scenarios, while discrepancies were present for TEFS. CONCLUSIONS: The LCAs passed all QA guidelines requirements for superficial hyperthermia delivery when used singularly or in an array configuration. A further characterization of parameters such as antenna efficiency and heat transfer coefficients would be beneficial for translating experimental results to simulated values. Implementing the QA guidelines was time-consuming and demanding, requiring careful preparation and correct setup of antenna elements.

Multi-echo gradient echo pulse sequences: which is best for PRFS MR thermometry guided hyperthermia?

PURPOSE: MR thermometry (MRT) enables noninvasive temperature monitoring during hyperthermia treatments. MRT is already clinically applied for hyperthermia treatments in the abdomen and extremities, and devices for the head are under development. In order to optimally exploit MRT in all anatomical regions, the best sequence setup and post-processing must be selected, and the accuracy needs to be demonstrated. METHODS: MRT performance of the traditionally used double-echo gradient-echo sequence (DE-GRE, 2 echoes, 2D) was compared to multi-echo sequences: a 2D fast gradient-echo (ME-FGRE, 11 echoes) and a 3D fast gradient-echo sequence (3D-ME-FGRE, 11 echoes). The different methods were assessed on a 1.5 T MR scanner (GE Healthcare) using a phantom cooling down from 59 °C to 34 °C and unheated brains of 10 volunteers. In-plane motion of volunteers was compensated by rigid body image registration. For the ME sequences, the off-resonance frequency was calculated using a multi-peak fitting tool. To correct for B0 drift, the internal body fat was selected automatically using water/fat density maps. RESULTS: The accuracy of the best performing 3D-ME-FGRE sequence was 0.20 °C in phantom (in the clinical temperature range) and 0.75 °C in volunteers, compared to DE-GRE values of 0.37 °C and 1.96 °C, respectively. CONCLUSION: For hyperthermia applications, where accuracy is more important than resolution or scan-time, the 3D-ME-FGRE sequence is deemed the most promising candidate. Beyond its convincing MRT performance, the ME nature enables automatic selection of internal body fat for B0 drift correction, an important feature for clinical application.

Display Characterization for Contrast Sensitivity Testing.

SIGNIFICANCE: Contrast sensitivity measurements using a computer display have been reported to lack accuracy when displaying small contrasts. This report investigates if the characterization/calibration of display luminance may contribute substantively to these kinds of described inaccuracies. PURPOSE: This study aimed to investigate what errors in contrast sensitivity may result from characterizing a display by fitting a gamma curve through physical or psychophysical luminance measurement data. METHODS: The luminance functions of four different in-plane switching liquid crystal displays (IPS LCDs) have been measured for all 256 gray levels (the actual luminance function). This has been compared with a gamma-fitted luminance curve (the gamma luminance function). Calculated are the errors in displayed contrast that may arise when assuming the gamma luminance function instead of the actual luminance function. RESULTS: The amount of error differs considerably between the displays. In general, for large contrasts (Michelson log CS <1.2), the error is acceptable (<<0.15 log unit). However, for smaller contrasts (Michelson log CS >1.5), the error may become unacceptably high (>0.15 log unit). CONCLUSIONS: To improve the accuracy of testing contrast sensitivity with an LCD, it is necessary to fully characterize the display, measuring the luminance of each gray level instead of fitting a smooth gamma function through limited luminance data.

Reciprocal c-di-GMP signaling: Incomplete flagellum biogenesis triggers c-di-GMP signaling pathways that promote biofilm formation.

The assembly status of the V. cholerae flagellum regulates biofilm formation, suggesting that the bacterium senses a lack of movement to commit to a sessile lifestyle. Motility and biofilm formation are inversely regulated by the second messenger molecule cyclic dimeric guanosine monophosphate (c-di-GMP). Therefore, we sought to define the flagellum-associated c-di-GMP-mediated signaling pathways that regulate the transition from a motile to a sessile state. Here we report that elimination of the flagellum, via loss of the FlaA flagellin, results in a flagellum-dependent biofilm regulatory (FDBR) response, which elevates cellular c-di-GMP levels, increases biofilm gene expression, and enhances biofilm formation. The strength of the FDBR response is linked with status of the flagellar stator: it can be reversed by deletion of the T ring component MotX, and reduced by mutations altering either the Na+ binding ability of the stator or the Na+ motive force. Absence of the stator also results in reduction of mannose-sensitive hemagglutinin (MSHA) pilus levels on the cell surface, suggesting interconnectivity of signal transduction pathways involved in biofilm formation. Strains lacking flagellar rotor components similarly launched an FDBR response, however this was independent of the status of assembly of the flagellar stator. We found that the FDBR response requires at least three specific diguanylate cyclases that contribute to increased c-di-GMP levels, and propose that activation of biofilm formation during this response relies on c-di-GMP-dependent activation of positive regulators of biofilm production. Together our results dissect how flagellum assembly activates c-di-GMP signaling circuits, and how V. cholerae utilizes these signals to transition from a motile to a sessile state.

Prototypical pacemaker neurons interact with the resident microbiota.

Pacemaker neurons exert control over neuronal circuit function by their intrinsic ability to generate rhythmic bursts of action potential. Recent work has identified rhythmic gut contractions in human, mice, and hydra to be dependent on both neurons and the resident microbiota. However, little is known about the evolutionary origin of these neurons and their interaction with microbes. In this study, we identified and functionally characterized prototypical ANO/SCN/TRPM ion channel-expressing pacemaker cells in the basal metazoan Hydra by using a combination of single-cell transcriptomics, immunochemistry, and functional experiments. Unexpectedly, these prototypical pacemaker neurons express a rich set of immune-related genes mediating their interaction with the microbial environment. Furthermore, functional experiments gave a strong support to a model of the evolutionary emergence of pacemaker cells as neurons using components of innate immunity to interact with the microbial environment and ion channels to generate rhythmic contractions.